Efavirenz (EFV) and nevirapine (NVP) are currently prescribed antiretroviral drugs of the same class that are hepatotoxic. Recently, a major role for cellular signaling pathways in modulating toxicity downstream of EFV metabolism has been reported in vitro using primary human hepatocytes. Increased oxidative stress and the upregulation of the proapoptotic protein BimEL (Bcl-2 interacting mediator of cell death extra long) have been demonstrated to be crucial in the stimulation of EFV-induced cell death, and NVP appears to modulate hepatocyte death via the same mechanism. The goal of this proposal is to determine the manner in which EFV and NVP upregulate BimEL, while also gaining a mechanistic understanding of the events downstream of BimEL that mediate the hepatocyte death. The aims are as follows: (1) Test whether genetic deficiency of proapoptotic BimEL results in protection against EFV- and NVP-mediated cell death in vivo: BimEL null mice will be used to determine whether the absence of BimEL renders mice resistant to hepatotoxicity stimulated by these compounds; RNA interference studies in hepatocytes will be used to determine the role of effector proteins, Bax and Bak, that are downstream of BimEL in modulating EFV- and NVP-induced hepatocyte death; siRNA and reporter gene assays will be used to define the mechanism by which EFV and NVP regulate the transcription of BimEL; (2) determine if proapoptotic pathways that are activated by antiretrovirals in primary human hepatocytes are also activated in macaques receiving antiretroviral therapy; (3) probe a role for alteration of the glutathione pathway in EFV- and NVP-induced hepatotoxicity; the effects of these compounds on the glutathione pathway will be examined using a mass spectrometry-based approach that will precisely quantitate the intermediates/products of this pathway; RNA interference will be used to define the role of enzymes in the glutathione pathway in EFV- and NVP-induced hepatotoxicity. It is expected that completion of the proposed studies will lead to the identification of novel therapeutic targets for the potential prevention and treatment of EFV- and NVP-mediated hepatotoxicity.